A large number of unique optical codes can be created with a class of rare earth based phosphor materials known as the PARALLUME™ materials as described below; These PARALLUME materials, which emit visible light upon excitation with either ultraviolet (UV) or infra red (IR) radiation, are excited at a single wavelength and subsequently emit several different colors of light into very narrow bands from several different rare earth emitting centers. A PARALLUME material contains one or more light-emitting species. The relative integrated emission intensities of two (2) or more light-emitting species (supplied by one or more PARALLUME materials) can be measured and represent an optical code as shown in FIGS. 1a-d. The UV→VIS process is referred to as a downconversion process as the higher energy UV is converted to lower energy visible light. The multicolor emitter materials that undergo this process are called the PARALLUME-UV materials. Another class of rare earth phosphors that contain ytterbium can convert near IR radiation into multicolored visible light by means of a multiphoton upconversion process and these materials are known as the PARALLUME-IR materials.
An optical barcode or unique optical signature can be generated using the PARALLUME materials by measuring the ratio of two different emitting species. The relative integrated emission intensities of two or more emitters within each PARALLUME composition can represent an optical barcode. For example, if a PARALLUME material contained two emitters, such as A and B, then a certain number of optical codes based on the relative emitting ratios of the two emitters can be resolved. If A and B could be optically resolved at 10% compositional intervals, then ten different compositions exist each of which represent a different ratio of the two emitters and therefore the system is said to have a multiplexing level of 10. The method by which the optical code is generated is by integrating the relative intensity of the emission peaks.
In practice, the code is always generated by analyzing the emission peak intensities in pairs of peaks to generate a ratio. For example, FIG. 2 shows a plot of the number of atoms of two emitters versus the ratio of the emission peak intensity from the two emitters.
There are two methods of synthesizing the PARALLUME materials so that two or more emitters are available to create the ratiometric based optical code. The two methods are to: (a) mix two or more emitters together to generate a known ratio by, for example, weighing various amounts of the two components or (b) synthesizing a single host material that contains known amounts of two or more emitters within the same crystallographic lattice. For the PARALLUME-UV downconverting compositions more precise definitions can be given for these two cases (a) and (b) directly above as:
(1) A downconverting composition comprising two or more lanthanide materials, wherein each lanthanide material comprises a host, an absorber, which can be the host itself, and one or more emitters, and wherein the materials emit detectable electromagnetic radiation upon excitation with absorbable electromagnetic energy, and wherein the emitted radiation is of a longer wavelength, or lower energy, than the absorbed radiation, and wherein one or more relative ratios of emission intensities uniquely identify the composition.
(2) A downconverting composition comprising a lanthanide material, wherein the lanthanide material comprises a host, an absorber which can be the host itself, and two or more emitters, and wherein the material emits detectable electromagnetic radiation upon excitation with absorbable electromagnetic energy, and wherein the emitted radiation is of a longer wavelength, or lower energy, than the absorbed radiation, and wherein one or more relative ratios of emission intensities uniquely identify the composition.
For the PARALLUME-IR materials, the compositions required to create ratios from the measurement of the integrated emission intensities of more than one emitter can be similarly prepared.
Because of the very narrow emission peak width of the rare earth centers in the PARALLUME materials, it is possible to measure the relative integrated intensity of the emission peaks with a very high degree of accuracy typically less than a percent coefficient of variance (% CV=standard deviation/mean×100) of less than 0.2-3%. Thus the multicolor emitting PARALLUME materials are capable of labeling and uniquely identifying millions of samples.
Many problems arise when developing a process to measure larger and larger numbers of samples but two of the more outstanding of these problems are (a) difficulties in labeling sufficiently large number of samples (insufficient multiplexing depth) because of the inability of the optical encoding system to generate a sufficient number of unique resolvable signatures and, (b) even if a method exists in principle to label and distinguish very large numbers of samples difficulties are often encountered in the logistics of actually synthesizing more than hundreds of samples.
An extremely high level of optical multiplexing has been previously demonstrated in the patent pending PARALLUME-UV and PARALLUME-IR materials with up to hundreds of thousands of optical codes in the PARALLUME-IR materials and billions of codes in the PARALLUME-UV materials statistically demonstrated. Since both of the commercially available optically encoded bead sets, such as those from Luminex and Quantum Dot (QD) Corporation, only have a multiplexing level of 100, this means that only 100 samples can be distinguished with a unique set of optical signatures before “running out” of unique codes. The current 100 level multiplexing limit is primarily due to the inherently broader emission peak widths in the organics dyes and quantum dots used to create the Luminex and QD optical codes and the fact that each dye or quantum dot has its own different excitation spectrum requiring multiple excitation sources for maximum brightness. The wider the emission peaks the fewer peaks can be resolved within a given wavelength range. While the relative integrated intensity of non-overlapping peaks can be obtained easily and accurately, as soon as emission peaks in a spectrum begin to display appreciable overlap the ability to integrate relative intensity is severely degraded. Since the PARALLUME optical codes are based on rare earth emitters, which have an inherently narrower peak width than the organic dyes or QDs, the PARALLUME materials can support a much deeper level of multiplexing.
Because the Luminex and QD systems have such a low level of multiplexing, practitioners of these technologies have never been faced with the daunting task of synthesizing tens or hundreds of thousands, or even millions, of different compounds. But in order to satisfy the synthetic requirements of a very deeply multiplexed system such as the PARALLUME-IR and PARALLUME-UV, the serially executed synthesis used for dozens of samples is totally inadequate and a hew method is required. Currently for example, one common way to prepare encoded beads is to entrain particles much finer than the final desired bead size within a polystyrene particle. The common magnetic beads are prepared by polymerizing styrene in the presence of appropriately sized particles of magnetite. But such serial methods would be totally inadequate for the numbers of samples required for large scale gene expression or genotyping experiments or for any sort of attribution or anticounterfeiting applications.
Therefore, when devising such a method for the fabrication of very large numbers of encoded beads or particles, the following requirements and attributes should be considered:
Synthesis Speed An examination of the number of syntheses required with respect to the time per synthesis (Table 1) shows that a very rapid synthesis method will be required. It is clear that normal serial synthesis methods will be totally inadequate to address the needs for highly parallel synthesis and that a new fabrication protocol is required.
TABLE 1Samples prepared within a given timeinterval at a given synthesis rateSample synthesis rateSamples/daySamples/yearOne/hour102000One/minute500100,000One/second30,0006,000,000
Accuracy of Synthesis Since the optical code is based on the ratios of the relative integrated intensities of the emission peaks of the various emitters present, and intensity of the emitted peaks is proportional to the amount of emitting material present, it is clear that the accuracy of constructing the requisite ratios must be carried out with an accuracy at least greater than the resolution interval to be determined. Since the particles to be labeled are generally in the range of 1 to 100 microns, the fabrication methodology must prepare ratios of the constituent PARALLUME materials very accurately using extremely small amounts of materials.
Reproducibility of Synthesis The reproducibility of the synthetic procedure must be appreciably better than the resolution interval to be determined when constructing the optical code. As with the accuracy of the synthesis discussed above the reproducibility must conform to the requirement that only miniscule amounts of PARALLUME material are present in each particle or bead.
Format Flexibility The optically encoded particles will more useful if the method used to fabricate the particles has the maximum flexibility in terms of such parameters as:
Number of particles Ideally the total number of particles, as well as the number of replicate particles, required for a given application could be controlled and be related to the number of variables or samples to be investigated. No extra particles need be prepared and all of the particles prepared would be used.
Particle Size The particle size must be compatible with the object to be labeled or identified. There are uses for labels in all size ranges. Larger labels can be prepared for compositions that are more difficult to read due to lack of brightness.
Particle Composition It would be desirable to have total compositional flexibility. By controlling the composition one can exactly match the optical multiplexing depth to the number of samples to be measured thereby spreading the code over the maximum compositional spread range and generating the easiest to read code.
There are two primary means by which particles are currently optically encoded depending on what the emitting species within the particles is. The two types are the organic dye molecule emitters, such as those found in the Luminex™ type products, and the quantum dot emitters from Quantum Dot Corporation™. The organic dye molecules are generally formed as a monomer with an appropriate functionality which becomes incorporated into a polymerizing system such as polystyrene during the formation of polystyrene beads. The quantum dot materials are either used directly or attached to other molecules or solids via an appropriate functional group. Alternatively, QD particles can be entrained in a system that is undergoing polymerization. For example, if a system consisting of small, monodisperse polystyrene beads is subjected to polymerization with, for example, styrene in the presence of the small QD particles (the QD particles are generally smaller than 10 nm in size), the small polystyrene bead will act as a site for the polymerization and the QD particles will be entrained in the polymerizing styrene. Thus the QD particles are trapped within the growing polystyrene bead. It should be noted that one of the primary difficulties with using either of these technologies is that both only provide for a 100 member bead set as of the time of this writing (2005). Therefore only 100 objects can be labeled and differentiated from one another in an experiment greatly limiting their utility as labels for sample numbers large enough to satisfy the requirements of high throughput experimentation.
A problem with both of these synthetic approaches is that the fabrication methods are incapable of producing millions of different codes within a very short period of time, e.g. less than 24 hours/million samples.
Accordingly, a method is needed for fabricating millions of different codes within a very short period of time and for encoding objects with such codes within such time periods.